HK1000143B - Lactic bacteria - Google Patents

Description

The purpose of the present invention is to culture a biologically pure strain of lactic acid bacteria, a composition containing this strain and a use of this strain.

EP 199535 (Gorbach and Goldin) proposes a bacterial strain identified first as Lactobacillus (L.) acidophilus and then as closest to L.casei subs. ramnosus (cf. M.Silva et al. in Antimicrobial Agents and Chemotherapy, 31, No 8, 1231-1233, 1987), which has good adhesion to cells of the small intestine lining and is suitable for therapeutic uses. This strain, called GG strain and registered with the ATCC (American Type Culture Collection) under No 53103, can be used in combination with an acidically acceptable pharmaceutical medium, particularly in food products, particularly in acidic dairy products such as yogurt.

Other strains of the same type have long been used in products and for similar purposes, but there is a need for particularly efficient strains of this type which are clearly identified, have undisputed virtues and enrich the range of available strains.

The present invention is intended to satisfy this need.

To this end, a biologically pure culture of a strain of lactic acid bacteria selected for its ability to implant in intestinal flora, to bind to intestinal cells, to competitively exclude pathogenic bacteria from intestinal cells, to immunomodulate and/or to reduce faecal enzyme activity is proposed.

Such a strain is particularly intended for use in humans or animals for therapeutic or prophylactic purposes in the gastrointestinal tract, in particular for anti-diarrheal purposes.

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The strain may also be administered as a composition including such culture and an unmanageable medium, in particular a pharmaceutically acceptable medium or a food product such as acidified milk, including yogurt or powdered milk formula, for example.

In a first preferred embodiment of the present invention, a culture of a strain of lactic acid bacteria selected for its ability to implant in the digestive tract of mice or rats with human gut flora is proposed.

In a second preferred embodiment of the present invention, a culture of a strain of lactic acid bacteria selected for its competitive ability to exclude, on intestinal cells, the pathogenic bacteria responsible for diarrhoea is proposed.

Among various strains of bacteria thus selected from acidified milk, in particular from commercial yoghurts, or from commercial crops intended for the preparation of such milk, or from the faeces of children, for example, four were deposited, as an example, under the Budapest Treaty, on 30.06.92 at the National Collection of Microorganism Cultures (CNCM), Institut Pasteur, 28 rue du Dr Roux, 75724 Paris Cedex 15, France, where they were each assigned the respective CNCM No. between parentheses, namely a strain of Lactobacillus acidophilus (CNCM I-1225), a strain of Bifidobacterium breve (IBCM-1226), a strain of Bifidobacterium infantis (CNCM I-1227) and a strain of Bifidobacterium long (CNCM I-1228).

Details of the morphology and general properties of these strains are given below:

The following shall be added to the list of active substances: Morphology of the blood:

Gram-positive, nonmotile, non-spore forming micro-organism.Sufficiently short and slender isolated rods

The metabolism:

Micro-aerophilic micro-organism with homofermentary metabolism leading to the production of lactic acid L ((+) and D ((-).Other characteristics: catalase (-), production of CO2 (-), hydrolysis of arginine (-).

Fermentation of sugars:

The active substance is a substance which is a mixture of amygdalin (+), arabinose (-), cellulose (+), esculin (+), fructose (+), galactose (-), glucose (+), lactose (+), maltose (+/-), mannitol (-), mannose (+), melibiose (-), raffinose (+), ribose (-), salicin (+), sucrose (+), trehalose (+).

Implantation in a gut flora Gnototoxic mice

Two groups of axonic mice (mice without gut flora) are each associated with human flora from a different donor (gnotoxic mice). After several days of installation, the gut flora of the mice is fully comparable to that of human donors, in functional, qualitative and quantitative terms.

In the present invention, many strains are tested for their ability to colonize the digestive tract of these mice with human flora, i.e. for their ability to implant in this gut flora.

It is found that most strains are not able to colonize these animals even after several successive inoculations, but that the strain L. acidophilus CNCM I-1225, for example, is able to multiply and implant itself in the digestive tract, i.e. in the intestinal flora of the mice of both groups, even after a single inoculation.

This colonisation or implantation allows the strain to be present in the faeces at a rate of more than 106 cfu/g. This stool content in viable strain germs may be considered necessary and/or sufficient for the metabolism of the strain to alter that of the host.

It is also noted that this implantation persists as long as the animals' environment is not disturbed.

Gnototoxic rats

Axein rats are associated (gnotoxic rats) with a strain (Bacteroides thetaiotaomicron FI 1, special collection of Nestec Research Centre SA, CH-1000 Lausanne, Switzerland) isolated from human gut flora from a healthy donor, which is intended, as described below, to simulate the production of enzymes from a complete faecal flora.

A test of L. acidophilus CNCM I-1225, for example, in this flora results in good co-colonisation which allows this strain to be also present in faeces at a rate of about 108 cfu/g.

Human volunteers

For comparison, the number of viable L. bulgaricus germs found in the faeces of healthy human volunteers consuming traditional yogurt, prepared by fermentation of cow's milk with a commercial culture of L. bulgaricus and S. thermophilus, is determined.

These volunteers ate no fermented dairy products for three consecutive periods of three weeks each, except for yogurt, which they ate during the second three-week period.

During the three weeks they ate yogurt, they did so in such a way that they ingested about 1010 L. bulgaricus per day, which is about 3 120 g yogurts per day.

The present invention involves a test in the same scenario as above, but with yogurt prepared by fermentation of milk with a culture of S.thermophilus and B.bifidus of commercial origin supplemented with the strain L.acidophilus CNCM I-1225, for example, in the same concentration.

The total number of viable Lactobacillus germs in the faeces of the volunteers before, during and after the period of consumption of yogurt is determined, with values of 105-106 cfu/g before, more than 107 cfu/g during and 106 cfu/g after.

The total number of Lactobacilli found in faeces during the period of yogurt consumption is therefore increased. The CNCM I-1225 strain was found in significant quantities and viable form in the volunteers. However, it was eliminated within a few days after stopping yogurt consumption.

Reduced faecal enzyme activity Gnototoxic rats

In the above tests with gnotobiotic rats, the activity of fecal azoreductase and nitroreductase was determined, as the enzymes azoreductase and nitroreductase are involved in the production of carcinogens, and high concentrations of these enzymes are associated with an increased risk of colon cancer.

The faecal enzymatic activity of Bacteroid-based gnotobiotic rats is found to be 2,5 μg/h/mg protein for azoreductase and 4,2 μg/h/mg protein for nitroreductase, whereas for Bacteroid-based gnotobiotic rats in whose flora, for example, CNCM I-1225 strain has been implanted, this enzymatic activity is 1,8 μg/h/mg protein for azoreductase and 3,5 μg/h/mg protein for nitroreductase.

Furthermore, it is found that gnobiotic rats with intestinal flora formed exclusively from CNCM strain I-1225 have no fecal azoreductase and nitroreductase activity.

In other words, the presence of CNCM strain I-1225 in the flora of gnotobiotic rats is found to induce a decrease in certain unwanted enzyme production in these animals, i.e. beneficial changes in the host metabolism.

Human volunteers

In the above human volunteer trials, the activity of the fecal nitro-reducing enzyme nitro-reducing is determined during the last days before the period of consumption of yoghurt prepared with CNCM I-1225, for example, during that period and in the first days afterwards.

It is thus found that this activity rises from 8.2 to 4.9 μg/h/mg of protein during the period of consumption of yogurt, remains at this level for about a week after this period and then gradually rises.

Immunomodulation Human volunteers (phagocytic power of leukocytes)

Human volunteers refrain from all consumption of fermented dairy products, except for products consumed according to the following schedule: milk for three weeks, yogurt prepared by fermentation of milk with a mixed culture of commercial S.thermophilus and L.acidophilus CNCM I-1225, for example, for the following three weeks, then milk for six weeks.

The phagocytic power of leukocytes in the peripheral blood of the volunteers is determined at the beginning and end of each of these periods.

This determination consists in extracting leukocytes from the blood and placing them in the presence of fluorescent bacteria. The fluorescent light emitted by leukocytes which have phagocytated fluorescent bacteria is measured by flow cytometric analysis (using a device marketed as Facscan). The percentage of leukocytes which have phagocytic activity is derived, which represents the said phagocytic power.

The peripheral blood leukocyte phagocytic potential is 36.5% at the beginning of the first period of milk consumption, 32.7% at the end of this period and therefore at the beginning of the period of yogurt consumption, 51.8% at the end of this period of yogurt consumption and 51.4% six weeks later, i.e. at the end of the second and last period of milk consumption.

Human volunteers (response to a vaccine)

16 healthy human volunteers (test group) were fed the following dietary regimen: for two weeks (weeks 1 and 2) a normal diet excluding any fermented product, for the following three weeks (weeks 3, 4 and 5) a diet with the consumption of three 125 ml yoghurts per day, these yoghurts being prepared by fermentation of milk with a commercial culture of S.thermophilus and Bifidobacterium bifidus to which was added, as an example, the strain of L.acidophilus CNCM I-1225 present in this yogurt at a rate of 107-108 cfu/ml, and for another two weeks (weeks 6 and 7) a normal diet excluding any fermented product.

14 healthy human volunteers (control group) are simultaneously fed a normal diet excluding any fermented products.

A vivotif oral vaccine (Salmonella typhi Ty21a) marketed by Berna SA is administered to volunteers in both groups as directed by the manufacturer on days 1, 3 and 5 of week 4.

Blood samples were taken from all volunteers 3 days before the start of week 3, and 1 day and 10 days after the end of week 5.

The specific IgA concentration of the immune response to Salmonella typhi lipopolysaccharide antigens (LPS) is determined by ELISA.

The increase in specific IgA concentration observed 15 days post-vaccination compared to 9 days prior to vaccination was significant in both groups (p < 0.001).

However, if we consider the ranges of increase factors < 2; > 2 and < 3; > 3 and < 4; > 4, we see distributions in these ranges of 1, 6, 3 and 6 volunteers for the test group versus 8, 3, 0 and 3 volunteers for the control group.

Adhesion to intestinal cells

The present invention investigates the adhesion of different strains of lactic acid bacteria to intestinal cells, including human Caco-2 intestinal epithelial cells (M. Pinto et al., Biol. Cell. 47, 323, 1983) and human HT29-MTX mucus secretory intestinal cells (Lesuffleur et al., Cancer Res. 50, 6334-6343) in monolayer culture in vitro.

To do this, cells are grown in 25 cm2 plastic vials (Corning) for cell line maintenance and on degreased, sterilized glass sheets (22x22mm) placed in six-well boxes (Corning) for adhesion testing.

To culture Caco-2 and HT29-MTX cells, the medium must be changed daily from the second day after re-seeding.

Lactic bacteria are grown anaerobically on MRS from a frozen stock.

A mixed incubation medium on the cells is prepared by mixing 50% of an antibiotic-free DMEM medium and 50% of the MRS medium in which the bacteria have grown, this medium containing 108 lactobacilli or bifidobacteria (cf. Chauvière G. et al., FEMS Microbiol.Lett.91, 213-218, 1992).

To achieve adhesion, the mixed medium containing the bacteria is placed on the intestinal cells and incubated for one hour in aerobic conditions. The well boxes are washed five times in twenty circular stirrings, so as to allow good removal of non-adherent bacteria. The cell mats are then fixed in successive methanol baths, 10 min to 70%, 10 min to 95% and 15 min to 100%, and coloured with Gram or Giemsa colouring.

Among the many strains tested, CNCM I-1225, has a good level of adhesion to intestinal cells as determined by these Caco-2 cell line adhesion tests.

Thus, the strain L. acidophilus CNCM I-1225 adheres to Caco-2 cells at a rate of about 150+23 bacterial cells per 100 Caco-2 cells.

For example, adherence tests on HT29-MTX cells of the L. acidophilus CNCM I-1225 strain have yielded even more spectacular results.

Surprisingly, the adhesion of this strain is also due to a factor it secretes in its own culture medium (e.g. MRS or milk), since if the one-hour incubation process on Caco-2 described above is carried out with L. acidophilus CNCM I-1225 strain without its bacterial culture medium, a significant decrease in adhesion is observed.

In addition, if this incubation process is carried out on Caco-2 with this strain and its culture medium previously treated with trypsin, a significant decrease in adhesion is also observed.

Competitive exclusion of pathogenic bacteria

The present invention investigates the competitive exclusion of pathogenic bacteria, including pathogenic bacteria responsible for diarrhoea, on intestinal cells by CNCM strain I-1225.

In particular, the exclusion of certain strains of E. coli, saprophytes of the digestive tract in humans and animals, which may become virulent and pathogenic, namely entero-toxinogenic E. coli (ETEC), entero-adherent E. coli (DAEC) and entero-pathogenic E. coli (EPEC), and the exclusion of one strain of Salmonella typhi-murium are being investigated.

The strains used for this study are: ETEC, strain H10407 which expresses CFA/1 (Collection of Prof. Joly, Laboratory of Microbiology, Faculty of Medicine and Pharmacy, University of Clermont-Ferrand 1, 63003 Clermont-Ferrand, France) for DAEC, strain C1845 (Collection of Dr. S.Bilge, Department of Microbiology, School of Medicine, G 3111 Health Sciences Building, University of Washington, Seattle, Washington 98195, USA) for EPEC, strain JPN15 pMAR7 which expresses EAF and eae (Collection of Prof. J.Kaper, Center for Vaccine Development, University of Maryland, School of Medicine, 10 South Pine Street, Maryland, 21201, USA) for Salmonella-murium, strain 1344 SL (School of Medicine, University of California, Stanford, School of Microbiology, Sauermilk and Immunology, Stanford, 33540-532, 33530-532, DSM, 33540-532, DSM, Stanford, USA)

To determine the adhesion of bacteria to Caco-2 cells, the following procedure is used: In short, the single layers of Caco-2 cells are washed twice with a saline phosphate (PBS) buffer. 14C-labelled E.coli or 35S-labelled Salmonella are suspended in the culture medium at 108 cfu/ml and 2 ml of suspension is added to each well containing a cell culture blade.

For E. coli, all incubations are conducted in the presence of 1% D-mannose. To determine an exclusion factor or rate, i.e. the proportion of pathogenic bacteria that are prevented from adhering to Caco-2 cells by lactic acid bacteria that take their place, 1 ml of suspension containing 108 cfu/ml of the marked pathogenic strain and 1 ml of suspension containing either 108 cfu/ml of the tested lactic acid strain are added to each pocket containing a cell culture plate.

The plates are incubated at 37°C in an atmosphere of 10% CO2 and 90% air for 1 hour. The cell monolayers are washed five times with sterile PBS. The adherent bacteria and intestinal cells are dissolved in NaOH 0.2N solution. The number of adherent bacteria marked by liquid scintillation count is evaluated.

Among different strains of lactic acid bacteria thus tested for their competitive exclusion or power of pathogenic bacteria, CNCM I-1225, selected and registered under the present invention, did indeed show remarkable capabilities as shown in the table below, which gives the percentage exclusion rates achieved by the tested strains at the expense of the different pathogenic strains used in these tests.

Souche (No CNCM)	Concentration (cfu/ml)	Taux d'exclusion compétitive (%), vis-à-vis de:
		ETEC	DAEC	EPEC	Salmonella
I-1225		78	79	83	86
		50	53	53	42
I-1226		80	68	83	88
		55	53	55	41
I-1228		47	47
		11
I-1227		58	46
		18

Claims (4)

1. Lactic bacterium having the registration number CNCM I-1225.
2. Food or pharmaceutical composition comprising the lactic bacterium having the registration number CNCM I-1225.
3. Composition according to claim 2, the said composition being an acidified milk or a powdered milk formula.
4. Use of the lactic bacterium having the registration number CNCM I-1225, in the preparation of a support which can be administered to man or to animals with therapeutic or prophylactic objectives in the gastro-intestinal system.